This document describes several methods for isolating and purifying DNA, RNA, and bacteriophages from plant and bacterial cells. For DNA isolation from plant cells, the method involves freezing and grinding plant tissue, lysing the cells with CTAB buffer, purifying the DNA with chloroform, precipitating it with isopropanol, washing it, and eluting the purified DNA. For plasmid and bacteriophage DNA isolation from bacterial cells, several techniques are described that separate DNA based on size or conformation differences, such as alkaline lysis and CsCl gradient centrifugation. RNA isolation methods include organic extraction to separate RNA from other cell components, as well as direct lysis methods.

1. DNA Isolation from Plant Cells
 Plant material (young green leaves) is frozen (freezing breaks cells) by using liquid nitrogen
and then ground with mortar and pestle to homogenize plant tissue.
Steps for DNA isolation from plant cells:
1. Lysis
 To 100 mg of homogenized tissue 500µl CTAB (cetyltrimethylammonium bromide)
extraction buffer is added and gently mixed by inversion.
 The sample is incubated at 65°C for 60 minutes in waterbath. Tubes are cooled to room
temperature. (CTAB is a cationic detergent which helps in cell lysis).
 EDTA (Ethylenediaminetetraacetic acid) is included in the extraction buffer to chelate
magnesium ions, a necessary co-factor for nucleases. As a result the nuclease activity is
inhibited due to lack of co-factor.
2. Purification of DNA
 After cooling the sample to room temperature, mixture of Chloroform: isoamyl alcohol
(24:1) is added and mixed by rocking the tube gently.
 Chloroform precipitates the cell components (carbohydrate, protein, and other contaminants).
Then the samples are centrifuged at 2,300 rpm for 2 minutes.
 After that the top aqueous layer (containing DNA) is transferred into a fresh tube and RNase
A is added. The sample is incubated for 30 minutes at room temperature (15-25°C).
3. Precipitation of DNA
 Isopropanol is added and mixed gently by inversion until a white fluffy DNA precipitate
appears (it should appear within 1 minute after addition of isopropanol).
 The sample is centrifuged at 2,300 rpm for 5 minutes at room temperature (15-25°C) and the
supernatant is discarded.

2. 4. Washing and Concentration of DNA
 Chilled CTAB Wash Buffer is added to the sample and mixed by pipetting. Sample is
incubated at room temperature for 20 minutes.
 After centrifuging the samples at 2,300 rpm for 5 minutes the supernatant is discarded. After
that cold 70% ethanol is added to the tube containing the DNA and mixed by pipetting.
 After that the sample is centrifuged at 2,300 rpm for 5 minutes. The supernatant is discarded.
The pellet is air dried for 10-15 minutes for the ethanol to evaporate.
5. Elution
 Elution Buffer is added and the pellet is resuspended.
Isolation of plasmid DNA
 A culture of cells, containing plasmids, is grown in liquid medium, harvested, and a cell
extract is prepared. In a plasmid preparation it is always necessary to separate the plasmid
DNA from the bacterial chromosomal DNA.
 The methods to separate plasmid DNA from genomic DNA are based primarily on two
differences: 1) size- plasmids are much smaller than bacterial DNA,
2) conformation- during preparation of the cell extract the chromosome is always broken
to give linear fragments while plasmid DNA remains circular.
(A) Separation on the basis of size
 Size fractionation is usually performed during preparation of the cell extract. If the cells
are lysed under very carefully controlled conditions, only a minimal amount of
chromosomal DNA breakage occurs. Cell disruption is carried out very gently to prevent
wholesale breakage of the bacterial DNA.
 The resulting DNA fragments are still much larger than the plasmids and can be removed
with the cell debris by centrifugation (
).

3.  For E. coli and related species, controlled lysis is performed by treatment with EDTA and
lysozyme.
 Lysis is carried out in the presence of sucrose, which prevents the cells from bursting
immediately. Instead, sphaeroplasts (cells with partially degraded cell walls but have an
intact cytoplasmic membrane) are formed.
 Cell lysis is now induced by adding a non-ionic detergent such as Triton X-100 (ionic
detergents, such as SDS, cause chromosomal breakage).
 After addition of detergent, centrifugation is done. It leaves a cleared lysate consisting
almost entirely of plasmid DNA.
Figure: Preparation of a cleared lysate.
 A cleared lysate retains some chromosomal DNA. Furthermore, if the plasmids
themselves are large molecules, they may also sediment with the cell debris. Size

4. fractionation is therefore rarely sufficient on its own, alternative ways of removing the
bacterial DNA contaminants must be considered.
(B) Separation on the basis of conformation
 Double-stranded DNA circles can take up one of two quite distinct configurations: 1) the
supercoiled conformation can be maintained only if both polynucleotide strands are intact,
hence the more technical name of covalently closed circular (ccc) DNA. 2) If one of
the polynucleotide strands is broken the double helix reverts to its normal relaxed state,
and the plasmid takes on the alternative conformation, called open-circular (oc).
 Most plasmids exist in the cell as supercoiled molecules. Supercoiling is important in
plasmid preparation because supercoiled molecules can be fairly easily separated from
non-supercoiled DNA. Two different methods are commonly used: 1) alkaline
denaturation and 2) ethidium bromide–caesium chloride density gradient centrifugation.
 Best results are obtained if a cleared lysate is first prepared.
1) Alkaline denaturation
 The basis of this technique is that there is a narrow pH range at which non-supercoiled
DNA is denatured, whereas supercoiled plasmids are not.
 If sodium hydroxide is added to a cell extract or cleared lysate, so that the pH is adjusted
to 12.0–12.5, then the hydrogen bonding in non-supercoiled DNA molecules is broken,
causing the double helix to unwind and the two polynucleotide chains to separate.
 If acid is now added, these denatured bacterial DNA strands reaggregate into a tangled
mass. The insoluble network is then pelleted by centrifugation, leaving plasmid DNA in
the supernatant.
 An additional advantage of this procedure is that, under some circumstances (specifically
cell lysis by SDS and neutralization with sodium acetate), most of the protein and RNA
also becomes insoluble and can be removed by the centrifugation step. Further
purification by organic extraction or column chromatography may therefore not be
needed if the alkaline denaturation method is used.

5. Figure: Plasmid purification by the alkaline denaturation method.
2) Ethidium bromide–caesium chloride density gradient centrifugation
This is a specialized version of the more general technique of equilibrium or density gradient
centrifugation.
Figure: Caesium chloride density gradient centrifugation. (a) A CsCl density gradient produced by
high speed centrifugation. (b) Separation of protein, DNA, and RNA in a density gradient.

6.  Density gradient centrifugation in the presence of ethidium bromide (EtBr) can be used
to separate supercoiled DNA from non-supercoiled molecules.
 Ethidium bromide binds to DNA molecules by intercalating between adjacent base pairs,
causing partial unwinding of the double helix.
 This unwinding results in a decrease in the buoyant density, by as much as 0.125 g/cm3
for linear DNA.
 However, supercoiled DNA does not have free ends therefore has very little freedom to
unwind, and can only bind a limited amount of EtBr.
 Therefore the decrease in buoyant density of a supercoiled molecule is much less, only
about 0.085 g/cm3. As a consequence, supercoiled molecules form a band in an
EtBr–CsCl gradient at a different position to linear and open-circular DNA.
 When a cleared lysate is subjected to this procedure, plasmids band at a distinct point,
separated from the linear bacterial DNA, with the protein floating on the top of the
gradient and RNA pelleted at the bottom.
 The position of the DNA bands can be seen by shining ultraviolet radiation on the tube,
which causes the bound EtBr to fluoresce. The pure plasmid DNA is removed by
puncturing the side of the tube and withdrawing a sample with a syringe.
Figure: Purification of plasmid DNA by EtBr–CsCl density gradient centrifugation

7.  The EtBr bound to the plasmid DNA is extracted with n-butanol and the CsCl removed by
dialysis. The resulting plasmid preparation is virtually 100% pure and ready for use as a
cloning vector.
Plasmid amplification
 Plasmids make up only a small proportion of the total DNA in the bacterial cell. The
yield of DNA from a bacterial culture may therefore be very low.
 The aim of amplification is to increase the copy number of a plasmid. Some multicopy
plasmids (those with copy numbers of 20 or more) have the useful property of being able
to replicate in the absence of protein synthesis.
 This contrasts with the main bacterial chromosome, which cannot replicate under these
conditions. This property can be utilized during the growth of a bacterial culture for
plasmid DNA purification.
 After a satisfactory cell density has been reached, an inhibitor of protein synthesis (e.g.,
chloramphenicol) is added, and the culture incubated for a further 12 hours.
 During this time the plasmid molecules continue to replicate, even though chromosome
replication and cell division are blocked. The result is that plasmid copy numbers of
several thousand may be attained. Amplification is therefore a very efficient way of
increasing the yield of multicopy plasmids.

8. Isolation of bacteriophage (λ) DNA
 For isolation of phage DNA the starting material is not normally a cell extract because
when cell extract of a phage infected bacterial culture is centrifuged, the bacteria are
pelleted and phage particles are left in suspension.
 The phage particles are then collected from the suspension and their DNA is extracted by
a single deproteinization step to remove the phage capsid.
 For obtaining satisfactory amount of phage DNA, the extracellular phage titer (the
number of phage particles per ml of culture) must be sufficiently high. Large culture
volumes, in the range of 500–1000 ml, are needed if substantial quantities of phage DNA
are required.
Preparation of non-lysogenic λ phages and growth of cultures to obtain a high λ titer
 The naturally occurring λ phage is lysogenic, and an infected culture consists mainly of
cells carrying the prophage integrated into the bacterial DNA.
 The extracellular λ titer is extremely low under these circumstances.
 To get a high yield of extracellular λ, the culture must be induced, so that all the bacteria
enter the lytic phase of the infection cycle, resulting in cell death and release of λ
particles into the medium.
 Strains of λ carrying a temperature-sensitive (ts) mutation in the cI gene are used for
this purpose.
 cI is one of the genes that are responsible for maintaining the phage in the integrated state.
If inactivated by a mutation, the cI gene no longer functions correctly and the switch to
lysis occurs.

9.  In the cIts mutation, the cI gene is functional at 30°C, at which temperature normal
lysogeny can occur. But at 42°C, the cIts gene product does not work properly, and
lysogeny cannot be maintained.
 A culture of E. coli infected with a λ phages carrying the cIts mutation can therefore be
induced to produce extracellular phages by transferring from 30°C to 42°C.
 The modified λ phages with deletion in cI and other genes cannot integrate into the
bacterial genome and can infect cells only by a lytic cycle.
Collection of phages from an infected culture
 The remains of lysed bacterial cells, along with any intact cells, can be removed from an
infected culture by centrifugation, leaving the phage particles in suspension.
 Phage particles are so small that they are pelleted only by very high speed centrifugation.
Collection of phages is therefore usually achieved by precipitation with polyethylene
glycol (PEG).

10.  PEG is a long-chain polymeric compound which, in the presence of salt, absorbs water,
thereby causing macromolecular assemblies such as phage particles to precipitate. The
precipitate can then be collected by centrifugation, and redissolved in a suitably small
volume.
Figure: Collection of phage particles by polyethylene glycol (PEG) precipitation
Purification of DNA from λ phage particles
 Deproteinization of the redissolved PEG precipitate is sometimes sufficient to extract
pure phage DNA, but usually λ phages are subjected to an intermediate purification step.
This is necessary because the PEG precipitate also contains a certain amount of bacterial
debris.
 These contaminants can be separated from the λ particles by CsCl density gradient
centrifugation. The λ particles band in a CsCl gradient at 1.45–1.50 g/cm3, and can be
withdrawn from the gradient.
 Removal of CsCl by dialysis leaves a pure phage preparation from which the DNA can
be extracted by either phenol or protease treatment to digest the phage protein coat.

11. Figure: Purification of λ phage particles by CsCl density gradient centrifugation.
Purification of M13 DNA
Double stranded form
 The double-stranded replicative form of M13, is very easily purified by the standard
procedures for plasmid preparation.
 A cell extract is prepared from cells infected with M13, and the replicative form is
separated from bacterial DNA by EtBr–CsCl density gradient centrifugation.
Single stranded form
 Steps required in single-stranded M13 DNA preparation involve: 1) growth of a small
volume of infected culture, 2) centrifugation to pellet the bacteria, 3) precipitation of the
phage particles with PEG, 4) phenol extraction to remove the phage protein coats, and 5)
ethanol precipitation to concentrate the resulting DNA.
 High titers of single-stranded form of the M13 genome, contained in the extracellular
phage particles are very easy to obtain. As infected cells continually secrete M13
particles into the medium, with lysis never occurring, a high M13 titer is achieved by
growing the infected culture to a high cell density.
 As the infected cells are not lysed, there is no problem with cell debris contaminating the
phage suspension.
 Consequently the CsCl density gradient centrifugation step, needed for λ phage
preparation, is rarely required with M13.

12. Figure: Preparation of single-stranded M13 DNA from an infected culture of bacteria
Isolation and Purification of RNA
 RNA (Ribonucleic acid) is a polymeric substance consisting of a long single-stranded
chain of phosphate and ribose units with the nitrogen bases adenine, guanine, cytosine
and uracil bonded to the ribose sugar present in living cells and many viruses.
 The steps for preparation of RNA involve homogenization, phase separation, RNA
precipitation, washing and re-dissolving RNA. The method for isolation and purification
of RNA are as follows:
1) Organic extraction method
2) Filter-based, spin basket formats
3) Magnetic particle methods
4) Direct lysis method
Organic extraction method
 This method involves phase separation by addition and centrifugation of a mixture of a
solution containing phenol, chloroform and a chaotropic agent (molecules that disrupt
non-covalent bonds) (guanidinium thiocyanate) and aqueous sample.
 Guanidium thiocyanate results in the denaturation of proteins and RNases, separating
rRNA from ribosomes.
 Addition of chloroform forms a colorless upper aqueous phase containing RNA, an
interphase containing DNA and a lower phenol-chloroform phase containing protein.

13.  RNA is collected from the upper aqueous phase by alcohol (2-propanol or ethanol)
precipitation followed by rehydration.
 One of the advantages of this method is the stabilization of RNA and rapid denaturation
of nucleases. Besides advantages, it has several drawbacks such as it is difficult to
automate, needs labor and manual intensive processing, and use of chlorinated organic
reagents.
Direct lysis methods
 This method involves use of lysis buffer under specified conditions for the disruption of
sample and stabilization of nucleic acids.
 If desired, samples can also be purified from stabilized lysates. This method eliminates
the need of binding and elution from solid surfaces and thus avoids bias and recovery
efficiency effects.
Advantages
• Extremely fast and easy.
• Highest ability for precise RNA representation.
• Easy to work on very small samples.
• Amenable to simple automation.
Drawbacks
• Unable to perform traditional analytical methods (e.g. spectrophotometric method).
• Dilution-based (most useful with concentrated samples).
• Potential for suboptimal performance unless developed/optimized with downstream
analysis.
• Potential for residual RNase activity if lysates are not handled properly.

14. Measurement of DNA concentration
 It is crucial to know exactly how much DNA is present in a solution when carrying out a
gene cloning experiment. Fortunately DNA concentrations can be accurately measured by
ultraviolet (UV) absorbance spectrophotometry.
 The purity of a solution of nucleic acid is determined by measuring the absorbance of the
solution at two wavelengths, usually 260 nm and 280 nm, and calculating the ratio of
A260/A280.
 Value of this ratio is 2.0 for pure RNA, 1.8 for pure DNA and 0.6 for pure RNA, DNA and
protein respectively.
 A ratio of less than 1.8 signifies that the sample is contaminated with protein or phenol and
the preparation is not proper.